Use Of A Fatty Acid For Preparing A Topical Composition For Allaying Inflammatory Reaction Due To Vennemous Hymenoptera Strings

ABSTRACT

The invention relates to the use of a fatty acid for preparing a composition for allaying inflammatory reactions due to hypenoptera stings.

The present invention relates to the use of a fatty acid for thepreparation of a topical composition for allaying inflammatory reactionsdue to hymenoptera stings.

Insects that secrete substances harmful to humans are rather numerous inour part of the world. Between wasps, hornets, bumblebees, ants andhoneybees, the risks of being stung are not insignificant. These insectsare called hymenoptera. Their sting can cause local or generalizednon-allergic reactions as well as local or generalized allergicreactions.

Local non-allergic reactions are due to a nonspecific inflammatoryreaction. A typical reaction to a hymenoptera sting consists ofimmediate pain combined with redness, itching and edema in an areaseveral centimeters in diameter. The progression of the reaction isvariable. The reaction can last a few hours (three on average) andgenerally does not last more than one day. Particular types of reactionsdo exist, however. Stings that lead to non-allergic reactions atspecific sites can have dramatic consequences. Stings in the oralcavity, in particular at the back of the throat, can lead to respiratorydisorders due to local edema. Finally, stings to the cornea of the eyecan cause immediate complications such as an ocular abscess orperforation of the globe. Certain complications may appear later,including cataracts or glaucoma.

Generalized non-allergic reactions may be encountered when multiplestings cause severe envenomation. These reactions include digestivedisorders with diarrhea and vomiting, a drop in blood pressureoccasionally associated with palpitations, sporadic convulsions and anattack on the muscles that can lead to renal failure (in the context ofrhabdomyolysis). These reactions generally appear after more thanapproximately thirty stings.

Allergic reactions concern roughly 1% of the population. They can belocal, regional, generalized or delayed. They are generally categorizedaccording to severity: local reaction (broader than the normal reactionbut limited to a limb), regional reaction (reaction that reaches thejoints of the stung limb), generalized reaction (distal skin,respiratory symptoms, Quincke's edema, asthma, digestive symptoms,palpitations), and anaphylactic shock (drop in blood pressure inaddition to the symptoms described, spontaneously fatal).

The hymenoptera include a number of species including bees, wasps,hornets and ants. Although most hymenoptera stings cause only moderatepain and a limited, temporary reaction, they may be dangerous, evenfatal, if multiple stings occur, if they occur in the mouth, in thethroat, or to the eye, or if the stung subject has an allergic reaction.The stung subject initially feels a more or less sharp pain depending onthe type of insect and the quantity of venom injected. The skin aroundthe sting becomes red and swollen. The subject feels itching that ismore or less intense.

Recent work has shown that insect venom, in particular bee venom, is acomplex substance comprised of a large number of active compoundsincluding histamine, melittin, hyaluronidase and phospholipase A.Sensitivity to these venom proteins may explain some of the reactionsdescribed above.

The applicant thus has found, surprisingly, that these fatty acids, inparticular C12-C24 acids, considerably reduce the inflammatory reactionthat follows a hymenoptera sting.

Thus, the present invention relates to the use of a fatty acid forpreparing a topical or injectable composition for allaying inflammatoryreactions due to hymenoptera stings.

The fatty acids used in the present invention are preferably naturalfatty acids, i.e., capable of being obtained from natural product suchas oils, but also synthetic fatty acids that are identical to or aredifferent from natural fatty acids.

Moreover, suitable fatty acids may be saturated or unsaturated fattyacids. The salts or the pharmaceutically acceptable derivatives of thesefatty acids that are also fatty acids may also be used.

Among the fatty acids used within the framework of the presentinvention, the following may be cited in particular: C12 to C24 acids,lauric (n-dodecanoic) acid, myristic (n-tetradecanoic) acid, palmitic(n-hexadecanoic) acid, stearic (n-octadecanoic) acid, arachidic(n-eicosanoic) acid, behenic (n-docosanoic) acid, lignoceric(n-tetracosanoic) acid, palmitoleic (cis-Δ⁹-hexadecenoic) acid, oleic(cis-Δ⁹-octadecenoic) acid, linoleic (cis,cis-Δ⁹-,Δ¹²-octadecadienoic)acid, linolenic (all-cis-Δ⁹-,Δ¹²-,Δ¹⁵-octadecatrienoic) acid, andarachidonic (all-cis-Δ⁵-,Δ⁸-,Δ¹¹-,Δ¹⁴-eicosatetraenoic) acid.

Polyunsaturated fatty acids are preferred, and linoleic acid and oleicacid are particularly active in the use according to the invention.

The fatty acids were tested in vitro for their capacity to inhibit theactivity of certain pro-inflammatory components of bee venom, and testedin vivo in rat in models of bee venom induced edemas.

The results of these tests show that fatty acids can be used to producecompositions for local treatment of inflammation resulting from venomoushymenoptera stings.

In the present case, the composition to which the invention relates mayalso include other active ingredients, notably an anesthetic and/or anantibiotic and/or an anti-allergic or anti-inflammatory substance.

The fatty acids can be formulated in any form suitable for topicaladministration, in combination with suitable excipients, to allowadministration of a dose of 0.01 mg to 50 mg per venomous hymenopterasting. Of course, the dose may vary according to the quantity of venomabsorbed, the number of stings or the type of insect.

Among the galenical formulations which may be suitable for implementingthe invention, ointments, creams, gels, patches, powders, sprays andlotions may be cited. Application via a stick may prove particularlyadvantageous for a single sting on a limb, for example. If the mucosa orthe eyes are stung, a formulation in the form of a spray or a collyrium,respectively, is preferred.

In certain cases it will be preferable to administer the compositionaccording to the present invention via local injection. The activesubstances of the pharmaceutical compositions according to the inventionmay be dissolved or suspended in a pharmaceutically-acceptable sterileinjectable liquid, such as sterile water, sterile organic solvent or amixture of these two liquids for local administration in a dose of 0.001mg to 1 mg per venomous hymenoptera sting.

The invention will be better understood upon consideration of theexamples below which refer to the following figures:

FIG. 1: Percentage of inhibition of bee venom PLA2 activity as afunction of acid concentration as follows:

FIG. 1A: Linoleic acid (example 1)

FIG. 1B: Oleic acid (example 1)

FIG. 2: Effect of oleic acid and linoleic acid on rat-paw volumeincrease in an inflammation model as a function of time following beevenom injection (example 2, experiment 1).

FIG. 3: Effect of oleic acid and linoleic acid on rat-paw volumeincrease in an inflammation model as a function of time following beevenom injection (example 2, experiment 2).

FIG. 4: Effect of oleic acid and linoleic acid on rat-paw volumeincrease in an inflammation model as a function of time following beevenom injection (example 2, experiment 3).

EXAMPLE I In vitro inhibiting effects of linoleic acid and oleic acid onbee venom phospholipase A2 (PLA2) activity, a major inflammatorycomponent of said venom.

The effect of linoleic acid and oleic acid on PLA2 activity is measuredaccording to the hexane extraction method described by M. Katsumata, G.Gupta, and A. S. Goldman, (1986), Anal. Biochem. 154 (2), 676-681. PLA2activity is determined by using a radioactive substrate,L-a-dipalmitoyl-[2,9,10(N)-3H-palmitoyl]-phosphatidylcholine. Thereaction is carried out in 1 ml of glycine/NaOH buffer, pH 9, containing2.2 mM deoxycholate, 0.11 μCi of dipalmitoyl-PC and 32 mU/ml of beevenom PLA2. After 20 minutes of incubation, the reaction is quenched byadding 0.2 ml of a Triton X-100/EDTA solution, and then the reactionproduct, radiolabeled palmitic acid, is extracted by a hexane solutioncontaining 0.1% acetic acid and 0.7 g/ml Na₂SO₄. The radioactivity ofthe extract is determined using a liquid scintillation counter. Theresults represent the mean±SEM of the CPM values obtained in twoindependent experiments.

FIGS. 1A and 1B illustrate the percentage of inhibition of PLA2 activity(vertical axis) as a function of the concentration of the fatty acidused (horizontal axis).

These results show that linoleic acid and oleic acid inhibit theenzymatic activity of bee venom PLA2 in a dose-dependent manner.

EXAMPLE II

Activity of oleic acid and linoleic acid in a rat model of inflammatoryedema caused by bee venom (G. A. Rabinovtch, E. C. Sotomayor, C. M.Riera, I. Bianco and S. G. Correa (2000), Eur. J. Immunol. 30,1331-1339; J. Chen, C. Luo, H. L. Li, and H. S. Chen, (1999), Pain, 83,67-76).

Experiment 1:

Bee venom (20 μg/ml in 0.9% NaCl) is incubated at room temperature inthe presence or absence of linoleic acid (32 μM). Thirty minutes afterincubation begins, the solutions (25 μl) are injected subcutaneously inthe upper surface of the foot of Sprague-Dawley rats (110-140 g). Edemais measured using a plethysmometer at 5, 10, 15, 20, 30, 60 and 90minutes following injection. The results represent the mean±SEM of 5animals. Statistical comparisons are performed using a t-test, for whichthe control group is comprised of animals having received venom alone.(*p<0.05, ***p<0.005) The results are represented in FIG. 2.

Experiment 2:

At 120 and 30 minutes before the injection of bee venom (20 μ0.9% NaCl),the Sprague-Dawley rats (110-140 g) either receive or do not receive, onthe top of the foot, an application of topical linoleic acid. Edema ismeasured using a plethysmometer at 5, 10, 15, 20 and 30 minutesfollowing subcutaneous injection of bee venom in the upper surface ofthe animal's foot. The results (FIG. 3) represent the mean+SEM of 20animals. Statistical comparisons are performed using a t-test, for whichthe control group is comprised of animals treated with the carrier(acetone).(*p<0.05, **p<0.01, ***p<0.005)

Experiment 3:

At 120 and 30 minutes before the injection of bee venom (20 μg/ml in0.9% NaCl), the Sprague-Dawley rats (110-140 g) either receive or do notreceive, on the top of the foot, an application of topical oleic acid.Edema is measured using a plethysmometer at 5, 10, 15 and 20 minutesfollowing subcutaneous injection of bee venom in the upper surface ofthe animal's foot. The results (FIG. 4) represent the mean±SEM of 5animals. Statistical comparisons are performed using a t-test, for whichthe control group is comprised of animals treated with the carrier(acetone). (*p<0.05)

1-6. (canceled)
 7. A method for allaying an inflammatory reaction due toa hymenoptera sting, comprising: administering a composition comprisinga fatty acid to a subject, wherein the composition is administered atleast one of topically or by injection.
 8. The method according to claim7, wherein the fatty acid comprises a C12-C24 acid.
 9. The methodaccording to claim 7, wherein the fatty acid comprises at least one oflauric (n-dodecanoic) acid, myristic (n-tetradecanoic) acid, palmitic(n-hexadecanoic) acid, stearic (n-octadecanoic) acid, arachidic(n-eicosanoic) acid, behenic (n-docosanoic) acid, lignoceric(n-tetracosanoic) acid, palmitoleic (cis-⁹-hexadecenoic) acid, oleic(cis-Δ⁹-octadecenoic) acid, linoleic (cis,cis-Δ⁹-,Δ¹²-octadecadienoic)acid, linolenic (all-cis-Δ⁹-,Δ¹²-,Δ¹⁵-octadecatrienoic) acid, orarachidonic (all-cis-Δ⁵-,Δ⁸-,Δ¹¹-,Δ¹⁴-eicosatetraenoic) acid.
 10. Themethod according to claim 7, wherein the fatty acid is selected from thegroup consisting of lauric (n-dodecanoic) acid, myristic(n-tetradecanoic) acid, palmitic (n-hexadecanoic) acid, stearic(n-octadecanoic) acid, arachidic (n-eicosanoic) acid, behenic(n-docosanoic) acid, lignoceric (n-tetracosanoic) acid, palmitoleic(cis-Δ⁹-hexadecenoic) acid, oleic (cis-Δ⁹-octadecenoic) acid, linoleic(cis,cis-Δ⁹-,Δ¹²-octadecadienoic) acid, linolenic (all-cis-Δ⁹-,Δ¹²-,Δ¹⁵-octadecatrienoic) acid, and arachidonic (all-cis-Δ⁵-,Δ⁸,Δ¹¹-,Δ¹⁴-eicosatetraenoic) acid.
 11. The method according to claim 7,wherein the fatty acid comprises at least one of linoleic acid or oleicacid.
 12. The method according to claim 7, wherein the fatty acid isselected from the group consisting of linoleic acid and oleic acid. 13.The method according to claim 7, wherein the composition has a form ofat least one of an ointment, a cream, a gel, a patch, a powder, a spray,a lotion or a stick.
 14. The method according to claim 7, wherein thecomposition is provided in an injectable form.
 15. The method accordingto claim 7, wherein the composition is applied to a region of tissueclose to the sting.